Stem Cell Cardomyocytes Used for Automated Patch Clamps

Inhibition of hERG cardiac ion channel must be tested for with any compounds in drug discovery in accordance with ICH guidelines. To be on the cautious side, it is often better to test compounds against a wider array of cardiac ion channels, e.g. hNaV1.5 and hCaV1.2 (Kramer et al., 2013).

Testing and additional ion channel targets are also demanded by the CiPA initiative; namely hNaV1.5 late current, hKir2.1, hKvLQT1 and Kv4.3 (Gintant et al., 2016).

These ion channel assays all respond to automated patch-clamp and are typically run using recombinant cell lines over-expressing an individual ion channel. The purpose of this research was to examine whether human-induced pluripotent stem cell-derived cardiomyocytes are a cost-effective and predictive cellular reagent for use on the QPatch automated patch-clamp system.

Methods

AXOL and Supplier B are the two vendors from which iPS-SC cardiomyocytes were purchased. The recommended proprietary medium was used to culture cells in accordance with the manufacturers recommended instructions. In brief, over 7-9 days, cells were thawed and cultured in fibronectin-coated T25 flasks.

On the day of assay, culture medium was added to stop the reaction after cells had been dissociated with Accumax for 20 minutes. After the addition of cold EC buffer, cells were incubated at 4 oC for 15 minutes, then assayed on QPatch HTX (Sophion). The extracellular and intracellular buffers used were (in mM):

EC1: NaCl 140, KCl, 4.5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, 5 μM blebbistatin, pH 7.45

EC2: NaCl 135, KCl 4.5, BaCl2 10, MgCl2 1, HEPES 10, glucose 10, 5 μM blebbistatin, pH 7.45

IC1: CsF 140, EGTA 1, HEPES 10, NaCl 10, 4 mM K2ATP, pH 7.25

IC2: KF 120, KCl 20, EGTA 10, HEPES 10, 4 mM K2ATP, pH 7.25

Protocols for standard QPatch cell positioning, sealing, and whole-cell access were used. In some experiments, the perforated patch was also used with 10 μM escin added to the IC buffer.

Voltage/current-clamp protocol:

Image Credit: Axol Bioscience

Results

Tests were carried out on iPS-SC cardiomyocytes from AXOL and Supplier B. Both cells cultured well and began to beat asynchronously after a few days in culture. AXOL cells beat at a frequency of 0.1-0.2 Hz and Supplier B at a frequency of 3-5 Hz. After 20 minutes of Accumax, cells dissociated completely. Sufficient cells were yielded in a T25 flask for two sequential experiments. It was possible to successfully store cells at 4 oC for up to one hour.

Cell positioning, sealing, and whole-cell access

Success rates for positioning, sealing, and whole-cell access of SC-CMs. Each experiment is a different cell preparation. 1-7 are Supplier B. 8-16 are AXOL.

Figure 1. Success rates for positioning, sealing, and whole-cell access of SC-CMs. Each experiment is a different cell preparation. 1-7 are Supplier B. 8-16 are AXOL. Image Credit: Axol Bioscience

Gaining whole-cell access was a limiting factor with SC-CMs. There were several unsuccessful attempts made to optimize QPatch whole-cell protocol parameters so the alternative was to use escin at 10 μM as a perforating agent. This too proved unsuccessful.

AXOL – Ion Channel Profile

As shown in Figure 2, several ionic currents were identified in SC-CMs from AXOL:

  1. In 53% of recordings (32/60) a verapamil-sensitive peak sodium current, ranging from 100 to 2000 pA was present
  2. In 84% of recordings made in Ba-containing EC2, a verapamil-sensitive barium current ranging from 100 to 2500 pA was present
  3. In several recordings, a ranolazine-sensitive late sodium current up to 200pA was present

Ionic currents recorded in cells from AXOL. A, peak sodium; B, barium; C, late sodium.

Figure 2. Ionic currents recorded in cells from AXOL. A, peak sodium; B, barium; C, late sodium. Image Credit: Axol Bioscience

In 52% of cells (31/60) action potentials were also recorded. As shown in Figure 3, these usually lasted 100-300 ms and were sensitive to verapamil. The action potentials were prolonged to >2000 ms in Ba2+-containing EC.

Action potentials recorded in cells from AXOL.

Figure 3. Action potentials recorded in cells from AXOL. Image Credit: Axol Bioscience

Even in the presence of the hERG activator (AZSMO-23), there were no hERG currents identified (Mannikko et al., 2015).

Supplier B – Ion Channel Profile

It was predominantly peak sodium currents recorded from these cells. These were expressed in 14% (3/21) cells and ranged from 50 to 400 pA. In 29% (6/21) cells action potentials were recorded.

What is the Outward Ppotassium Current?

A slow-to-activate, outward potassium current was identified, as large as 2000 pA when recording with potassium-containing IC2 buffer, at +40 mV. There were inconsistencies in the time course of activation in comparison to cardiac IKs current.

Image Credit: Axol Bioscience

Conclusions

  • The beating frequency and ion channel profile differ in SC-CMs from different suppliers.
  • Our results suggest that SC-CMs from AXOL express cardiac sodium and calcium channels, hNaV1.5 and hCaV1.2, respectively.
  • Firing action potentials are also possible with SC-CMs.
  • Approximately 40% of experiments resulted in successful whole-cell recordings on the QPatch. The increase in success rate was insignificant with attempts at optimization.
  • Voltage and current-clamp are combined in the same sweep with the QPatch, making it a useful and novel platform for working with SC-CMs.

SC-CMs are heterogenous, making them a challenging and expensive reagent to use on automated patch-clamp. However, they do express significant and relevant cardiac ion channels.

References and Further Reading

  • Kramer et al. (2013) MICE Models: Superior to the HERG Model in Predicting Torsade de Pointes. Scientific Reports 3:2100
  • Gintant et al. (2016) Evolution of strategies to improve preclinical cardiac safety testing. Nat Rev Drug Disc 15:457-471
  • Mannikko et al. (2015) Pharmacological and electrophysiological characterization of AZSMO-23, an activator of the hERG K(+) channel. BJP 172: 3112-25

Acknowledgments

Produced from materials originally authored by Michael Morton and Lauren Eades from ApconiX Ltd.

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Feb 18, 2020 at 11:31 AM

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